Human plasminogen is a single-chain protein containing 791 amino acid residues. Activation of plasminogen to plasmin results from a single cleavage of the Arg561-Val562 peptide bond in the zymogen. The resulting plasmin molecule is a two-chain, disulfide-linked serine protease with trypsin-like specificity (cleaves after Lys and Arg).
The amino-terminal heavy chain of plasmin (residues 1-561, ˜60 kDa) is composed of five kringle domains, each containing approximately 80 amino acid residues. The kringle domains are responsible for the regulatory properties of plasminogen, such as interaction with activation inhibitors, e.g., Cl−1 ions; with activation stimulators, e.g., ε-aminocaproic acid; with mammalian and bacterial cells; and with other proteins, such as plasmin physiological substrate fibrin and plasmin inhibitor α2-antiplasmin. Of all five kringles, kringle 1 is one of the most multi-functional: its lysine-binding activity has been shown to be responsible for plasmin interaction with α2-antiplasmin and fibrin. See Wiman, B., et al., Biochim. Biophys. Acta 579:142-154 (1979); and Lucas, Mass., et al., J. Biol. Chem. 258:4249-4256 (1983).
The C-terminal light chain of plasmin (residues 562-791, ˜251 kDa) is a typical serine protease, homologous to trypsin and containing the classic serine protease catalytic triad: His603, Asp646, and Ser741. Plasminogen contains 24 disulfide bridges and 2 glycosylation sites, on Asn289 and Thr346.
The limited proteolysis of plasminogen by elastase has been shown to result in three fragments (Sottrup-Jensen, L., et al., Prog. Chem. Fibrinol. Thrombol., 3:191-209 (1978)). First fragment, K1-3, includes the first three kringles and can be isolated in two versions, Tyr79-Val338 and Tyr79-Val354. The second fragment, K4, corresponds to the fourth kringle and includes residues Val355-Ala440. The last, C-terminal fragment (the so-called mini-plasminogen) includes residues Val443-Asn791 and consists of the fifth kringle and the serine protease domain. Mini-plasminogen can be activated in the same way as plasminogen, forming mini-plasmin.
Because of the complex structure of the full-length plasminogen molecule, bacterial expression systems have not proven useful for recombinant plasminogen production. Plasminogen is produced in the form of insoluble inclusion bodies and is not re-foldable from that state. Further, the expression of plasminogen in mammalian cells is complicated by intracellular activation of plasminogen into plasmin and the resulting cytotoxicity. Production of fully active plasminogen using insect cells is possible, however, this system is not suitable for large-scale production due to low yield.
Accordingly, a modified recombinant protein, possessing the desirable characteristics of plasmin/plasminogen while lacking certain negative characteristics and being capable of production in bacterial cells in substantial quantities, is desirable.